Nucleic acid sequencing and analysis, as well as other types of macromolecular separations, are often performed by electrophoresis in slab gels. Many of these procedures use gradient gels as a means of increasing sensitivity, range and versatility.
Polyacrylamide gels with a gradient in porosity are prepared by varying the concentration of acrylamide in one direction along the gel. The variation permits the gel to be used for fractionating a mixture of species with molecular weights spanning a broad range. Depending on the suspected composition of the mixture, the gradient can be linear, exponential, or specifically tailored to conform to any selected profile. Separations can be performed with the gradient either parallel or perpendicular to the direction of electrophoretic migration, although most of these separations are performed with a parallel gradient.
Gels containing a denaturant are used in certain separations of nucleic acids, particularly DNA sequencing procedures. A gradient of the denaturant is often used as a means of differentiating among sequences to distinguish fragments of mutant genes from those of wild-type genes. The partial melting, or strand dissociation, of a double-stranded DNA fragment due to exposure to denaturing conditions is sequence-dependent, with certain domains of the double strand dissociating (and thus forming single-strand loops) more readily than others. These discrete melting domains within the fragment cause a retardation in the electrophoretic mobility of the fragment as a whole. The retardation is therefore related in a complex way to the base sequence of the fragment. By applying the DNA, whether mutant or wild type, in a continuous line across one edge of the slab gel and using a denaturing gradient perpendicular to the direction of migration, each fragment will form an S-shaped curve, indicating a drop from a relatively fast migration rate to a relatively slow rate at the denaturant concentration at which the least stable domain of the fragment melts. The inflection points of these curves will be distributed along the gradient, with the S-curves and hence the fragments being readily differentiated on the basis of their base sequences rather than their molecular size.
Gels with denaturing gradients parallel to the direction of migration are also used, notably as the second stage of a two-dimensional separation. Separation along the first dimension is usually a conventional constant gel separation, where the differentiation is on the basis of molecular size. Separation along the second dimension then differentiates on the basis of sequence. Using multiple lanes in the first, constant-gel stage, the lane containing the separated fragments is removed and placed along one edge of the gradient gel at the end of the gradient containing the lowest denaturant concentration. As the fragments migrate into high-denaturant concentrations, they separate into subfragments according to their base sequences.
Depending on the size of the gel and the type of separation being performed, the ease with which a gradient gel can be formed depends on the dimensions of the slab. The slabs are frequently of much greater length than width, and both length and width are several orders of magnitude greater than the depth of the slab. Slabs such as these are generally formed between two glass plates with thin spacer strips along the side edges of the plates, separating the plates by their thickness, and thereby establishing the thickness of the gels held in between. This arrangement makes it difficult in some cases to inject gelforming solution of continuously varying composition in such a manner that the incoming liquid will not mix and thereby obscure the gradient sought to be formed. In addition, for perpendicular gradient gels, the gel must be cast in one direction, then rotated 90.degree. to run the electrophoresis. The present invention addresses these problems by providing a means of injecting gel-forming solution through the spacer strips in such a manner that the solution can be injected into the space with the enclosure rotated on its side so that the spacer strips are essentially horizontal, and the enclosure then rotated back to the upright position to engage the electrophoresis cell for sample preparation.